Molecular characterization of the β‐lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh,Saudi Arabia

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β‐lactamases such as metallo‐β‐lactamases (MBL) and extended spectrum β‐lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β‐lactamases. Here, the prevalence of different β‐lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β‐lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi‐resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates.     

Exploration of Genetic Variations through Single‐cell Whole‐genome Sequencing in the Model Ciliate Tetrahymena thermophila

Ciliates are unicellular eukaryotes with separate germline and somatic genomes and diverse life cycles, which make them a unique model to improve our understanding of population genetics through the detection of genetic variations. However, traditional sequencing methods cannot be directly applied to ciliates because the majority are uncultivated. Single‐cell whole‐genome sequencing (WGS) is a powerful tool for studying genetic variation in microbes, but no studies have been performed in ciliates. We compared the use of single‐cell WGS and bulk DNA WGS to detect genetic variation, specifically single nucleotide polymorphisms (SNPs), in the model ciliate Tetrahymena thermophila. Our analyses showed that (i) single‐cell WGS has excellent performance regarding mapping rate and genome coverage but lower sequencing uniformity compared with bulk DNA WGS due to amplification bias (which was reproducible); (ii) false‐positive SNP sites detected by single‐cell WGS tend to occur in genomic regions with particularly high sequencing depth and high rate of C:G to T:A base changes; (iii) SNPs detected in three or more cells should be reliable (an detection efficiency of 83.4–97.4% was obtained for combined data from three cells). This analytical method could be adapted to measure genetic variation in other ciliates and broaden research into ciliate population genetics.     

Co-production of Tet(X) and MCR-1, two resistance enzymes by a single plasmid

Tigecycline and colistin are few of ‘last-resort’ antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.     

Current and emerging polymyxin resistance diagnostics: A systematic review of established and novel detection methods

The emergence of polymyxin resistance, due to transferable mcr genes, threatens public and animal health as there are limited therapeutic options. As polymyxin is one of the last-line antibiotics, there is a need to contain the spread of its resistance to conserve its efficacy. Herein, we describe current and emerging polymyxin resistance diagnostics to inform faster clinical diagnostic choices. A literature search in diverse databases for studies published between 2016 and 2020 was performed. English articles evaluating colistin resistance methods/diagnostics were included. Screening resulted in the inclusion of 93 journal articles. Current colistin resistance diagnostics are either phenotypic or molecular. Broth microdilution is currently the only gold standard for determining colistin MICs (minimum inhibitory concentration). Phenotypic methods comprise of agar-based methods such as CHROMagar™ Col-APSE, SuperPolymyxin, ChromID^® Colistin R, LBJMR and LB medium; manual MIC-determiners viz., UMIC, MICRONAUT MIC-Strip and ComASP Colistin; automated antimicrobial susceptibility testing systems such as BD Phoenix, MICRONAUT-S, MicroScan, Sensititre and Vitek 2; MCR-detectors such as lateral flow immunoassay (LFI) and chelator-based assays including EDTA- and DPA-based tests, that is, combined disk test, modified colistin broth-disk elution (CBDE), Colispot, and Colistin MAC test as well as biochemical colorimetric tests, that is, Rapid Polymyxin NP test and Rapid ResaPolymyxin NP test. Molecular methods only characterize mobile colistin resistance; they include PCR, LAMP and whole-genome sequencing. Due to the faster turnaround time (≤3 h), improved sensitivity (84%–100%) and specificity (93.3%–100%) of the Rapid ResaPolymyxin NP test and Fastinov^®, we recommend this test for initial screening of colistin-resistant isolates. This can be followed by CBDE with EDTA or the LFI as they both have 100% sensitivity and a specificity of ≥94.3% for the rapid screening of mcr genes. However, molecular assays such as LAMP and PCR may be considered in well-equipped clinical laboratories.     

Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐lactamases in Southern India

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP‐PCR) and multi‐locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA‐carbapenemases, metallo‐β‐lactamases (MBLs) and efflux pumps. REP‐PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103^B. Second most prevalent ST belonged to clonal complex (CC) 92^B which is also referred to as international clone II. Most of the isolates were multi‐drug resistant, being susceptible only to polymyxin‐B and newer quinolones. Class D β‐lactamases such as bla_{OXA‐51‐like} (100%), bla_{OXA‐23‐like} (56.8%) and bla_{OXA‐24‐like} (14.8%) were found to be predominant, followed by a class B β‐lactamase, namely bla_IMP‐1 (40.7%); none of the isolates had bla_OXA‐58 like, bla_NDM‐1 or bla_SIM‐1. Genes of efflux‐pump adeABC were predominant, most of isolates being biofilm producers that were PCR‐positive for autoinducer synthase gene (>94%). Carbapenem non‐susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA‐type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.     

MiR‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.     

Identification of de novo germline mutations and causal genes for sporadic diseases using trio‐based whole‐exome/genome sequencing

Whole‐genome or whole‐exome sequencing (WGS/WES) of the affected proband together with normal parents (trio) is commonly adopted to identify de novo germline mutations (DNMs) underlying sporadic cases of various genetic disorders. However, our current knowledge of the occurrence and functional effects of DNMs remains limited and accurately identifying the disease‐causing DNM from a group of irrelevant DNMs is complicated. Herein, we provide a general‐purpose discussion of important issues related to pathogenic gene identification based on trio‐based WGS/WES data. Specifically, the relevance of DNMs to human sporadic diseases, current knowledge of DNM biogenesis mechanisms, and common strategies or software tools used for DNM detection are reviewed, followed by a discussion of pathogenic gene prioritization. In addition, several key factors that may affect DNM identification accuracy and causal gene prioritization are reviewed. Based on recent major advances, this review both sheds light on how trio‐based WGS/WES technologies can play a significant role in the identification of DNMs and causal genes for sporadic diseases, and also discusses existing challenges.     

Arabidopsis thaliana GLUTATHIONE‐S‐TRANSFERASE THETA 2 interacts with RSI1/FLD to activate systemic acquired resistance

A partly infected plant develops systemic acquired resistance (SAR) and shows heightened resistance during subsequent infections. The infected parts generate certain mobile signals that travel to the distal tissues and help to activate SAR. SAR is associated with epigenetic modifications of several defence‐related genes. However, the mechanisms by which mobile signals contribute to epigenetic changes are little known. Previously, we have shown that the Arabidopsis REDUCED SYSTEMIC IMMUNITY 1 (RSI1, alias FLOWERING LOCUS D; FLD), which codes for a putative histone demethylase, is required for the activation of SAR. Here, we report the identification of GLUTATHIONE‐S‐TRANSFERASE THETA 2 (GSTT2) as an interacting factor of FLD. GSTT2 expression increases in pathogen‐inoculated as well as pathogen‐free distal tissues. The loss‐of‐function mutant of GSTT2 is compromised for SAR, but activates normal local resistance. Complementation lines of GSTT2 support its role in SAR activation. The distal tissues of gstt2 mutant plants accumulate significantly less salicylic acid (SA) and express a reduced level of the SA biosynthetic gene PAL1. In agreement with the established histone modification activity of FLD, gstt2 mutant plants accumulate an enhanced level of methylated and acetylated histones in the promoters of WRKY6 and WRKY29 genes. Together, these results demonstrate that GSTT2 is an interactor of FLD, which is required for SAR and SAR‐associated epigenetic modifications.     

Identification and characterization of a fusarium head blight resistance gene TaACT in wheat QTL‐2DL

Fusarium head blight (FHB) resistance in wheat is considered to be polygenic in nature. Cell wall fortification is one of the best resistance mechanisms in wheat against Fusarium graminearum which causes FHB. Metabolomics approach in our study led to the identification of a wide array of resistance‐related (RR) metabolites, among which hydroxycinnamic acid amides (HCAAs), such as coumaroylagmatine and coumaroylputrescine, were the highest fold change RR metabolites in the rachis of a resistant near‐isogenic line (NIL‐R) upon F. graminearum infection. Placement of these metabolites in the secondary metabolic pathway led to the identification of a gene encoding agmatine coumaroyl transferase, herein referred to as TaACT, as a candidate gene. Based on wheat survey sequence, TaACT was located within a FHB quantitative trait loci on chromosome 2DL (FHB QTL‐2DL) between the flanking markers WMC245 and GWM608. Phylogenetic analysis suggested that TaACT shared closest phylogenetic relationship with an ACT ortholog in barley. Sequence analysis of TaACT in resistant and susceptible NILs, with contrasting levels of resistance to FHB, led to the identification of several single nucleotide polymorphisms (SNPs) and two inversions that may be important for gene function. Further, a role for TaACT in FHB resistance was functionally validated by virus‐induced gene silencing (VIGS) in wheat NIL‐R and based on complementation studies in Arabidopsis with act mutant background. The disease severity, fungal biomass and RR metabolite analysis confirmed TaACT as an important gene in wheat FHB QTL‐2DL, conferring resistance to F. graminearum.     

Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes

Colistin resistance has attracted substantial attention after colistin was considered as a last-resort drug for the treatment of infections caused by carbapenem-resistant and/or multidrug-resistant (MDR) Gram-negative bacteria in clinical settings. However, with the discovery of highly mobile colistin resistance (mcr) genes, colistin resistance has become an increasingly urgent issue worldwide. Despite many reviews, which summarized the prevalence, mechanisms, and structures of these genes in bacteria of human and animal origin, studies on the prevalence of mobile colistin resistance genes in aquaculture and their transmission between animals and humans remain scarce. Herein, we review recent reports on the prevalence of colistin resistance genes in animals, especially wildlife and aquaculture, and their possibility of transmission to humans via the food chain. This review also gives some insights into the routine surveillance, changing policy and replacement of polymyxins by polymyxin derivatives, molecular inhibitors, and traditional Chinese medicine to tackle colistin resistance.     

Development and comparison of SYBR Green quantitative real‐time PCR assays for detection and enumeration of sulfate‐reducing bacteria in stored swine manure

Aims: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real‐time PCR detection of sulfate‐reducing bacteria (SRB) in stored swine manure. Methods and Results: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR‐R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus‐like Group 1 SRB in manure. Conclusions: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.